Members of the RNA virus family Hypoviridae persistently alter phenotypic traits and attenuate virulence of their pathogenic fungal host, primarily by disrupting cellular signal transduction pathways. Consequently, hypoviruses provide utility as biological control agents and as unique tools for identifying fungal virulence determinants and designing antimycotic therapeutic strategies. Recent efforts to understand how hypoviruses attenuate fungal virulence have provided additional insights into the role of cellular G-protein signaling in fungal virulence and resulted in the discovery of a novel component of G-protein signal transduction. The functional activity domain of a known hypovirus-encoded symptom determinant was mapped in detail. Multiple new virus-encoded modifiers of host phenotype were mapped through the construction of recombinant chimeric hypoviruses derived from infectious cDNA clones of mild and severe hypovirus isolates. Advantage was also taken of these chimeras to demonstrate the feasibility of engineering hypoviruses to fine-tune the interaction between a pathogenic fungus and its host. Efforts to exploit hypoviruses as gene vectors resulted in the unexpected identification of several dispensable and essential elements of viral replication. Four new Specific Aims have been designed to capture the full advantage of newly developed experimental approaches for further elucidation and exploitation of hypovirus molecular biology while meeting the criteria of feasibility, relevance to ongoing studies and general significance. These include i) focused characterization of hypovirus ORF B polyprotein processing, ii) detailed mapping of hypovirus symptom determinants using recombinant chimeric hypoviruses, iii) detailed characterization of mapped symptom determinants and iv) further characterization of recently identified dispensible and essential replication elements. The results derived from completion of these aims will have immediate relevance to ongoing efforts to enhance the utility of hypoviruses and other potentially efficacious RNA viruses for purposes of manipulating their hosts in novel and productive ways.